Hindawi

BioMed Research International

Volume 2017, Article ID 1436080, 12 pages
https://doi.org/10.1155/2017/1436080

Review Article
Novel Treponema pallidum Recombinant Antigens for
Syphilis Diagnostics: Current Status and Future Prospects

Aleksey Kubanov, Anastassia Runina, and Dmitry Deryabin

Department of Laboratory Diagnostics of Sexually Transmitted Diseases and Dermatoses,
State Research Center of Dermatovenereology and Cosmetology, Korolenko Street 3/6, Moscow 107076, Russia

Correspondence should be addressed to Dmitry Deryabin; dgderyabin@yandex.ru

Received 2 February 2017; Accepted 21 March 2017; Published 24 April 2017

Academic Editor: György Schneider

Copyright © 2017 Aleksey Kubanov et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

The recombinant protein technology considerably promoted the development of rapid and accurate treponema-specific laboratory
diagnostics of syphilis infection. For the last ten years, the immunodominant recombinant inner membrane lipoproteins are proved
to be sensitive and specific antigens for syphilis screening. However, the development of an enlarged T. pallidum antigen panel for
diagnostics of early and late syphilis and differentiation of syphilis stages or cured syphilis remains as actual goal of multidisciplinary
expertise. Current review revealed novel recombinant antigens: surface-exposed proteins, adhesins, and periplasmic and flagellar
proteins, which are promising candidates for the improved syphilis serological diagnostics. The opportunities and limitations of
diagnostic usage of these antigens are discussed and the criteria for selection of optimal antigens panel summarized.

1. Treponema pallidum Biology
Treponema pallidum belongs to the family Spirochaetaceae,
order Spirochaetales, phylum Spirochaetes, which is a phylo-
genetically ancient and distinct group of bacteria. Due to the
cell structure, physiology, genetics, and pathogenic features T.
pallidum is a very unusual microorganism [1].
T. pallidum is a Gram-negative spiral-shaped bacterium,
which varies in length from 5 to 15 𝜇m and is 0,20 𝜇m in
diameter. T. pallidum is covered with the outer membrane
(OM), periplasmic space with endoflagella, peptidoglycan
layer, and inner membrane (IM), which surrounds a cytoplas-
mic cylinder [2]. Three to six flagella extend in periplasmic
space from both ends toward the centre of microorganism
and determine the helical shape and characteristic corkscrew
motility (rotating around longitudinal axis) of T. pallidum
cells. This motility allows T. pallidum to permeate through
membranous and gel-like substances and is important for
T. pallidum invasion and dissemination during the syphilitic
infection.
T. pallidum is a microaerophilic bacterium with an
optimal growth temperature of 37∘ C and minimal metabolic
capabilities. The microorganism is able to carry out glycolysis
and interconversion of amino acids and fatty acids but
lacks tricarboxylic acid cycle and alternative carbon sources
pathways; de novo synthesis of amino acid, fatty acid, and
nucleotides or enzyme cofactors synthesis pathways are also
absent [3]. As a result, T. pallidum utilizes most of the essential
molecules and substrates from the host environment using
numerous specific transporters and does not survive outside
the mammalian host [4]. T. pallidum is a strictly extracellular
pathogen, which is in a direct contact with the humoral
and cellular immunity mechanisms and therefore can evade
elimination and migrate into the immunologically privileged
tissues of the host organism.
In fact, fastidious nature of this bacterium is a probable
result of its long-term evolution and adaptation to the host
environment [5], and that made T. pallidum one of the most
dangerous human pathogens since 1495 till the development
of antibiotic therapy.
2. Treponema pallidum Genome
A reference strain of T. pallidum subsp. pallidum is Nichols,
which was isolated in 1912 from the cerebrospinal fluid of
syphilitic patient in Washington DC and then was cultivated
2 BioMed Research International
in rabbit testes. The novel reference strain, which is pheno-
typically distinct from Nichols stain, is Street Strain 14 (SS14),
isolated for the first time in 1977 in Atlanta from the skin
lesion of a patient with secondary syphilis.
In 1998 T. pallidum Nichols strain became one of the first
annotated bacterial genomes using Sanger sequencing [6]. In
2008, the genome of the SS14 strain was sequenced by an
oligonucleotide array [7]. In 2013 both T. pallidum Nichols
and SS14 strains were resequenced using next-generation
sequencing, and that considerably improved the genome
annotation [8].
The genome assembly showed a single circular chro-
mosome of 1,138,006 bp or 1,139,633 bp with a G + C base
composition of 52.8% and a total of 1,041 or 1,039 predicted
open reading frames (ORFs) in Nichols and SS14 strains,
respectively. About 5% of T. pallidum genes are specific
to the family Spirochaetaceae, whereas most of them are
genus- and species-specific. Of the 1,041 ORFs in T. pallidum
Nichols strain, only 577 (55% of total) have predicted biologic
functions based on sequence similarities, while 177 ORFs
(17%) match hypothetical proteins and 287 ORFs (28%) have
no database match and may be novel genes. Among the 1,039
ORFs in genome of T. pallidum SS14 strain, functions of
444 genes (43%) were not determined also [9]. Recently, the
function of 207 hypothetical proteins was predicted using
sequence- and structure-based method and was hypothesized
for more 237 genes.
In summary, these results showed one of the smallest
bacterial genomes (only few intracellular pathogenic species
have a smaller genome) and greatly stimulated the study of
this uncultivable in vitro bacterium [10].
3. Treponema pallidum Proteome
The identification of T. pallidum proteins began in 1975
with the application of electrophoretic techniques. Based on
the SDS-PAGE results the T. pallidum protein pattern was
described and the nomenclature was firstly standardized [11].
This format consists of the prefix TpN (for T. pallidum Nichols
strain) followed by a consensus relative molecular mass value.
Further, two-dimensional gel electrophoresis (2DGE) tech-
nique significantly improved T. pallidum proteome research.
In a prominent study of McGill et al. [12] 2DGE was used
for T. pallidum Nichols strain analyses, which in accordance
with amino acid sequence data showed highly expressed
proteins. In spite of more than 1000 expected proteins only
148 spots that represented 88 polypeptides were identified by
their relative positions in 2DGE patterns, which were high-
level expressed genes products.
A recent T. pallidum proteome characterization using
complementary mass spectrometry technique revealed 557
unique proteins at a high level of confidence, including 106
items firstly accounted at the protein level [13]. These data
provide most valuable insights into in vivo T. pallidum protein
expression representing 54% of the predicted proteome.
The unusual feature of the T. pallidum proteome sub-
cellular location was an extremely low density of proteins
located in the outer membrane (approximately 1% of the
number found in the E. coli outer membrane) [14]. OM
proteins include the species-specific family of 12 T. pallidum
repeat (Tpr) proteins, and some of them were predicted to be
involved in membrane permeability. Other surface-exposed
proteins were firstly designated as Treponema spp. rare outer
membrane proteins (TROMPs), while newly identified OM
proteins began to denote Tp prefix followed by ORF number
in T. pallidum genome (e.g., Tp0326, Tp0453), and this format
is now the most common.
Located in periplasmic space flagellar proteins (tradi-
tionally denoted as Fla) are widely presented in T. pallidum
proteome, namely, FlaB1, FlaB2, and FlaB3 proteins of the
spiral filament inner core covered with FlaA protein of the
outer sheath, complemented with hook-associated and IM
located complex of flagellar motor proteins that are typical
for Spirochaetaceae family [15].
A recent analysis of the T. pallidum proteome predicted
the presence of a large number of lipoproteins and also a
high-level expression of lipoprotein genes [16]. Most of them
are located in the inner membrane, where they play a role in
nutrient reception and transport or have unpredicted functions.
A proposed allocation of T. pallidum proteins in trepone-
mal cell membrane is presented in Figure 1.
4. Treponema pallidum Immunoproteome
The set of proteins, which induced immune response in the
host and showed reactivity with sera from syphilis patients,
was termed as T. pallidum immunoproteome. In prominent
Brinkman et al. [17] and McGill et al. [12] studies that investi-
gated protein expression library and T. pallidum strain Nichols
proteins extracted from testicular tissue of infected rabbits,
respectively, only 34–38 reactive antigens were detected.
There is no complete identity between these sets of proteins
(Figure 2), however principal proteins matched together.
Firstly, some inner membrane lipoproteins were typically
reactive with sera from patients at all stages of syphilis, and
this subset of seroreactive antigens strictly correlates with
Brinkman et al. and McGill et al. immunoproteome studies.
Despite the fact that lipoproteins are not exposed on the bac-
terial surface and are preliminarily located in IM, they are able
to induce the high-level immune response, so these were the
antigens to use for syphilis diagnostics, and that significantly
developed the treponema-specific serological tests.
Secondly, few surface-located antigens were revealed in T.
pallidum immunoproteome, and that reflects the extremely
low density of proteins in the outer membrane of this
microorganism. The small number of these potential targets
limits the overall detection of the spirochete and allows the
pathogen to evade the host immune system, giving its name
a “stealth” pathogen [18]. This subset differ in Brinkman
et al. and McGill et al. immunoproteome studies, and still
OM proteins play a significant role in the outer membrane
permeability and adhesion to biopolymers and T. pallidum
antigenic variability, and that determines current interest to
their role in pathogenicity and their diagnostical use.
Taken together, the immunoproteome data suggest the
low T. pallidum immunogenicity and also allow specifying the
most promising antigens for syphilis diagnostics.
BioMed Research International 3

Tp0326

Outer Tp0117 Tp0663 (Tp92)

membrane (Tromp2)
(TprC)

Tp0453
Periplasmic
space

FlaA1/FlaB
Endoflagella

Peptidoglycan layer

Tp0435 Tp0768
(Tpp17) (TmpA)

Tp0684 Tp0319 Tp0171

Tp0574 Tp0821
(MglB) (TmpC) (Tpp15) (Tp47) (Tp32)

Glu
Pcn Met
Gal

Inner
membrane

Cytoplasm

Figure 1: Topological model of T. pallidum seroreactive (lipo)proteins proposed localization.

5. Treponema pallidum Recombinant expensive due to the inability of T. pallidum to be cultured in

Lipoproteins as a Source for
Sensitive and Specific Serological
Diagnostics of Syphilis
The initial treponema-specific tests (i.e., assays for detection
of specific antibodies) used native or sonically disrupted T.
pallidum cells as the source of total number of antigens for the
fluorescent treponemal antibody-absorbance test, T. pallidum
particle agglutination, and T. pallidum hemagglutination
assay [19]; however, this approach was very complicated and
vitro.
Recently the recombinant protein technology promoted
considerably the development of treponema-specific labora-
tory diagnostics [20]. Most typically the T. pallidum DNA
derived from Nichols strain genome is amplified by PCR and
inserted into an expression vector and then to Escherichia coli
cells for expression of fusion proteins with a tag sequence for
efficient chromatography purification. Then obtained recom-
binant proteins were tested as antigens in either enzyme-
linked immunosorbent assay (ELISA) or Western blot (WB)
format. This approach greatly changed the knowledge of
4 BioMed Research International

Recombinant protein ELISA Tp15-Tp17-Tp47 was established as an instrument for rapid,

2D-PAGE and simple, and convenient syphilis serological screening in
Tp0100 Tp0693 immunoblotting the clinical setting using diagnostic ELISA method [30] or
Tp0133 Tp0727 miniaturized protein biochip technique [31].
Tp0136 Tp0750 In some other studies the subset of recombinant lipopro-
Tp0435
Tp0225 Tp0767
Tp0030 Tp0584 teins, which induce the strongest antibody response and
Tp0768 Tp0056 Tp0605 are currently used in T. pallidum diagnostic tests, includes
Tp0257 Tp0772
Tp0574 Tp0108 Tp0608 44.5 kDa lipoprotein (TmpA; tp0768 gene product) [32] and
Tp0277 Tp0789
Tp0292 Tp0821
Tp0122 Tp0747 chimeric E. coli expressed Tpp15-Tpp17-Tp44.5-Tp47 antigen
Tp0171 Tp0748 (Meridian Life Science, Inc., Memphis, Tennessee USA).
Tp0326 Tp0954 Tp0163
Tp0249 Tp0792 More rarely the recombinant products of tp0319 gene (TmpC,
Tp0327 Tp0956 Tp0216
Tp0259 Tp0844 35 kDa purine nucleoside receptor lipoprotein) [33] and
Tp0398 Tp0971 Tp0319 Tp0349 Tp0862 tp0684 gene (MglB-2, methylgalactoside ABC transporter,
Tp0463 Tp0974
Tp0684 Tp0365 Tp0868 41 kDa homolog of galactose/glucose-binding lipoprotein)
Tp0470 Tp0993
Tp0769 Tp0400 Tp0870 [34] are also used [35, 36] preliminarily in Western blot assay
Tp0486 Tp1015
Tp0971 Tp0426 Tp0886 as a combination of these immunogenic antigens [37] and
Tp0625 Tp1016
Tp1038 Tp0453 Tp0921 are proved to be highly sensitive and specific for acquired
Tp0663
Tp0505 Tp0925 syphilis.
Tp0509 Tp0965 In recent observation the Tp32 lipoprotein (tp0821
gene product) was characterized as L-methionine-binding
lipoprotein localized in T. pallidum inner membrane [38].
Figure 2: T. pallidum proteins, which exhibit immunoreactivity
Despite the fact that this protein was low reactive in
with serum from syphilis patients in Brinkman et al. 2006 (recombi- Brinkman et al. immunoproteome study [17] and was not
nant protein ELISA) and McGill et al. 2010 (2D-PAGE immunoblot- detectably reactive in McGill et al. study [12] the serological
ting) proteome research. Bold indicates proteins discussed in the tests based on Tp0821 showed 91,0% and 98,3% positive rates
present review. of the IgM ELISA and the IgG ELISA, respectively, which
correlated with the results of other treponemal tests [38].
The specificity was 94,3–100% when Tp0821 immunoassay
syphilis immunology and together with immunoproteome was cross-checking with serum samples obtained from 30
research led to the selection of optimal antigen combinations patients with Lyme disease, 5 patients with leptospirosis, and
for T. pallidum serological detection [21]. 52 uninfected controls. According to these data the authors
Several strong immunogenic antigens that induced a indicated Tp0821 as a new diagnostic antigen that requires
high antibody response during syphilis infection and are not further verification and confirmation.
cross-reactive with serum from patients with other spiro- In summary, treponema-specific tests based on the tech-
chetal diseases have been identified. In this set the 15 kDa nology of recombinant lipoproteins significantly improved
lipoprotein (Tp15; tp0171 gene product) and the major outer the diagnostics of syphilis providing an excellent 95–99% sen-
membrane 17 kDa lipoprotein (Tp17; tp0435 gene product) sitivity and specificity of serological assays, being less effective
are the key members [22]. At the moment the function in early and late stages of syphilis diagnostics [21] and in
of Tp15 is still unknown, while Tp17 is characterized as estimating the effect of therapy. Also, they do not distinguish
an eight-stranded 𝛽-barrel protein with a shallow “basin” between disease stages, while immunoproteome researches
at one end of the barrel and an 𝛼-helix stacked on the indicate the possibility of differential immune response to
opposite end [23], which probably plays a role in either certain T. pallidum antigens during infection. Thus, the devel-
protein ligand binding, treponemal membrane architecture opment of enlarged recombinant antigen panel for T. pallidum
maintenance [24], or syphilis pathogenesis by activation of detection remains as actual goal of multidisciplinary molec-
the expression of intercellular adhesion molecule 1 (ICAM-1), ular biology, microbiology, and immunology research [39].
E-selectin, and monocyte chemoattractant protein-1 (MCP-1)
genes in endothelial cells [25]. Another strong immunogen 6. Surface-Exposed Treponema pallidum
is 47 kDa lipoprotein (Tp47; tp0574 gene product) which Proteins for Improved Serological
is a carboxypeptidase (major T. pallidum penicillin-binding Diagnostics of Syphilis
protein) [26] and plays a role in host-pathogen interaction
via stimulation of microvessel endothelial cells to synthesize The OM proteins are the most important immunological
intercellular adhesion molecule and via induction of vascular targets due to their availability in intact T. pallidum cells;
cell adhesion molecule [27]. however, they are rare compounds. In Cox et al. study
The early syphilis diagnostics were based on a single [14] only 17 candidates for OM proteins were identified: 7
recombinant antigen, where sensitivities and specificities of members of the Tpr family, 9 non-Tpr hypothetical proteins,
Tp15, Tp17, and Tp47 were 100% and 96%; 100% and 100%; and TP0326 (Tp92).
100% and 20%, respectively [28], while assays with two or The specific T. pallidum repeat (Tpr) family of proteins
three antigen combinations resulted in the improvement of includes 12 members, which can be divided into three
diagnostic assay [29]. Further an artificial fusion lipoprotein subfamilies [40]. Subfamily I includes genes tprC, tprD,
BioMed Research International
tprI, and tprF; and subfamily II includes genes tprE, tprG,
and tprJ which encode products with common N- and C-
termini flanking central domains that differ in sequence
and length, while less homologic subfamily III (tprA, tprB,
tprH, tprK, and tprL) differs in variable regions. Subfamily
I Tpr proteins [41] possess a conserved sequence at the N-
and C-termini and central regions and are predicted to be
located in the outer membrane and involved in membrane
permeability. For example, the TprC/D (Tp0117/0131) and
TprI [42] are proposed to be trimeric, pore-forming proteins
with identical 𝛽-barrels and N-terminal periplasmic domains
that directly or indirectly link the barrels to the peptidoglycan
layer (Figure 1). An extensively studied TprK (tp0897) of
the III subfamily genes undergoes variation of seven variable
regions (V1–V7) by nonreciprocal recombination with a large
repertoire of “donor sites” to generate new mosaic proteins
[43]. Because the V regions are recognized as the significant
targets of the humoral immune response, it can be considered
that immune selection of new TprK variants is a mechanism
for “antigenic shift” of T. pallidum immune evasion and
persistence [44].
Surprisingly, despite the strong antibody and T-cell
responses against the N-terminal conserved region of the
subfamily I Tpr proteins and TprK protein, the Tpr proteins
were not indicated as seroreactive in both Brinkman et al. and
McGill et al. immunoproteome researches [12, 17]. Probably,
this may be determined by the above-described Tpr proteins
variability, since TprK sequences differ substantially between
and within individual strains, and, as a result, patients
often contain multiple T. pallidum clones expressing different
variants of the tprK gene [45]. According to these facts the
Tpr family is hypothesized to be essential for T. pallidum
pathogenesis and evasion from the host immune system,
making it a longstanding objective to further vaccine research
but limiting its importance as diagnostic antigens.
The group of non-Tpr surface-exposed treponema rare
outer membrane proteins, designated as TROMPs, includes
three members: TROMP-1 (31-kDa), TROMP-2 (28-kDa),
and TROMP-3 (65-kDa) proteins. In previous studies [46]
TROMPs were shown to be antigenic when tested with
serum from infected rabbits and humans; however, in
Brinkman et al. immunoproteome research [17] TROMP-2
(FlaA homolog, tp0663 gene product) was only reactive with
sera from primary-syphilis patients, and in McGill et al. study
[12] this protein was identified in T. pallidum proteome by
2DGE-MS phoresis but was not found to be reactive with
human sera in immunoblot analysis. Recently, recombinant
Tp0663 protein was confirmed to be a new serodiagnostic
candidate antigen, which was extremely sensitive (98.83%)
and specific (100%) for the detection of all stages of the
syphilis infection [47].
Another surface-exposed Tp0326 protein (Tp92; tp0326
gene product) was described by Cameron et al. [48], using
a differential screening strategy to identify E. coli clones
expressing T. pallidum opsonic targets, and later it was
characterized as BamA (𝛽-barrel assembly machinery pro-
tein A) ortholog with the sequence homology to a known
Gram-negative family of highly conserved 𝛽-barrel com-
pounds [49]. Structural modeling of Tp0326 predicted five
5
polypeptide transport-associated (POTRA) domains in the
N-terminus and 18-stranded amphipathic 𝛽-barrel in the
C-terminus, which are responsible for the native protein’s
amphiphilicity [50] (Figure 1). According to Kenedy et al.
study related to Tp0326 protein BB0795 of Borrelia burgdor-
feri (Lyme disease spirochete) its function is essential for
the assembly of OM proteins [51]. Tp0326 is seroreactive
in Brinkman et al. immunoproteome [17], while it did not
exhibit reactivity with early latent syphilis sera and did
not show immunogenicity in McGill et al. study [12]. This
observation can be explained by extremely low level of
Tp0326 expression in T. pallidum proteome as variation in the
antibody responses to POTRA and 𝛽-barrel portions of this
antigen. Surprisingly, T. pallidum infected rabbits exhibited
an antibody response to both antigenic epitopes, whereas
humans with secondary syphilis respond to POTRA only
[49]. Recently, Luthra et al. [50] showed that only the 𝛽-
barrel domain of Tp0326 contains surface-exposed epitopes
in intact T. pallidum and identified an immunodominant
large L4 extracellular loop. Based on these results the syphilis
diagnostic tests and kits based on Tp0326 recombinant
protein, its combination with Tp0453 antigen, and Tp0326-
0453 chimeric fusions were developed [52].
Tp0453 (tp0453 gene product) is a 287 a.a. protein
(putative lipoprotein) associated with the inner surface of T.
pallidum outer membrane (Figure 1). In Hazlett et al. study
[53] this nonlipidated variant of the protein exhibited exten-
sive 𝛽-sheet structure and amphipathic 𝛼-helices, whereby
when added to artificial bilayers it showed multiple mem-
brane inserting and enhanced the membrane permeability,
suggesting being a porin. More recently, the 3D crystal struc-
ture of Tp0453 has been solved. It consists of a 𝛼/𝛽/𝛼-fold
and includes five stably folded amphipathic helices, which
are crucial for Tp0453 integration into the membrane [54].
Based on structural dynamics and Mycobacterium tubercu-
losis lipoproteins’ comparison data, Tp0453 was proposed to
be a carrier of lipids and glycolipids during outer membrane
biogenesis. Resuming, Tp0453 is hypothesized to be a novel
type of bacterial outer membrane protein, which may render
the T. pallidum outer membrane permeability to nutrients
while remaining inaccessible to antibodies.
Tp0453 is not seroreactive in Brinkman et al. immuno-
proteome research [17], but in McGill et al. study it showed
a moderate seroreactivity and was found to be preliminarily
reactive with sera from primary-syphilis patients [12]. This
observation correlated with Van Voorhis et al. data [55],
which exhibited 100% specificity and sensitivity for Tp0453
in reaction with syphilis patients’ sera, giving negative result
with relapsing-fever, Lyme disease, or leptospirosis patients’
sera. Recently, the methods of producing soluble recombinant
Tp0453 via expression in pET28a vector were developed and
kits including the soluble and solid substrates, containing
Tp0453 protein, are also provided. The development of
ELISA method using Tp0453 recombinant products and
chimeric Tp0453-Tp0326 proteins as diagnostic antigens are
reported. The sensitivities of Tp0453 and the Tp0453-Tp0326
chimera were found to be 98% and 98%, respectively, and
the specificities were 100% and 99%, respectively, which
characterized these proteins as novel candidate antigens for
6 BioMed Research International
treponema-specific serological diagnostics [52]. Now Tp0453
together with conventional Tp15, Tp17, Tp47, TmpA, and
novel Tp0257 (Gpd) antigens is included into the panel
of commercially available WB kit Recom Blot Treponema
IgG/IgM 2.0 (Mikrogen GmbH, Germany).
7. Novel Treponema pallidum Derived
Recombinant Products: Adhesins
and Periplasmic and Flagellar
Proteins—Opportunities and Limitations
Tp0155 and Tp0483 were predicted as two putative adhesins
of the T. pallidum genome and then demonstrated spe-
cific attachment to fibronectin and blockage of a bacterial
adherence to fibronectin-coated slides [56]. Interestingly,
Tp0155 preferentially binds to the matrix form of fibronectin,
whereas Tp0483 binds to both the soluble and matrix forms,
which exist in different conformational forms with cryptic
epitopes becoming exposed during fibronectin matrix assem-
bly. Recently Tp0155 was described as a protein comprising
a leader peptide, two N-terminal LysM domains, which rec-
ognize carbohydrate polymers, and M23 peptidase sequence,
which makes this protein able to degrade peptidoglycan and
exhibit the enzymatic activity [57]. In turn, the analyses of
Tp0483 outer membrane protein revealed two fibronectin
binding regions between 274–289 and 316–333 amino acids
residues [58]. In addition to the adhesive and enzymatic
functions both T. pallidum proteins induce production of IL-
6, IL-1𝛽, and TNF-𝛼 in macrophages, and that is associated
with the activation of NF-𝜅B [59].
Surprisingly, these proteins were not seroreactive in
immunoproteome researches, and in comparative study with
Gpd both Tp0155 and Tp0483 [55] gave positive result with
only 9% of syphilis patient sera, and all of these reactive sera
were from the individuals with early primary infection.
Tp0136 is 485 a.a., 49 kDa hypothetical protein/lipopro-
tein exposed on the T. pallidum outer membrane. The recom-
binant protein study revealed the ability to bind fibronectin
and laminin glycoproteins, which involved Tp0136 attached
to the host extracellular matrix components [60]. Recently it
was shown that Tp0136 adheres more efficiently to cellular
than to plasma fibronectin via its N-terminal conserved
region [61]. Additionally, Tp0136 is highly transcribed during
an experimental infection in parallel with the host immune
response to the pathogen, which suggests a possible role for
this protein in T. pallidum persistence.
TP0136 is not reactive in McGill et al. proteome; however
in Brinkman et al. study this protein exhibits reactivity
to human sera compared to rabbit sera [17] preliminarily
with primary-syphilis stage. Recently, the Tp0136 selective
fragment (Tp0136B) with a molecular weight of about 28 kDa
was tested with sera from primary-syphilis patients, and the
positive result was shown in 85.5% of cases [62].
Tp0751 firstly was described as 237 a.a., 25,8 kDa protein
and then was identified as T. pallidum laminin-binding
adhesin [63], which is crucial for pathogen dissemina-
tion in the host organism due to the attachment to the
extracellular matrix component laminin—major glycopro-
tein found within mammalian basement membrane. The
laminin-binding region in Tp0751 is limited to 10-amino acid
fragment, and this motif inhibited the attachment of T. pal-
lidum to laminin, as well as Tp0751-specific antibodies inhibit
the attachment of T. pallidum to laminin too. Further stud-
ies showed the Tp0751 bifunctionality including fibrin clot
degradation capability and characterized this molecule as the
treponemal metalloprotease pallilysin [64]. Cotranscribed
protein Tp0750 was described as a serine protease, which
degrades major clot components (fibrinogen and fibronectin)
[65], and was hypothesized to work in cooperation with
Tp0751 and together to play a role in T. pallidum invasion and
dissemination in the host organism.
Despite the significance for T. pallidum pathogenicity,
Tp0751 is not seroreactive in both abovementioned immuno-
proteome researches, while Tp0750 exhibited a week serore-
activity in Brinkman et al. study with sera from primary
and early latent syphilis [17]. Finally, in comparative studies
Tp0751 revealed lower seroreactivity than Tp0257 (Gpd) and
Tp1038 (TpF1) [55]. However, being limited for diagnostic
use, the Tp0751 protein showed good immunoprotective
properties, allowing it to be considered as a promising
syphilis vaccine candidate [66].
Firstly Tp0257 protein was identified as a potential
immunoreactive antigen using a differential immunologic
expression library screening. According to the results of
nucleotide sequence analysis this protein was demon-
strated to be the 356-residue homologue of glycerophos-
phodiester phosphodiesterase (Gpd) [67], an enzyme, that
hydrolyzes deacylated phospholipids to alcohol and glycerol-
3-phosphate, previously identified in Haemophilus influen-
zae, Escherichia coli, Bacillus subtilis and Borrelia hermsii. The
characterization of the recombinant protein Tp0257 showed
its bifunctionality, revealing both the enzymatic activity and
the capability of binding the Fc-fragment of human IgA,
IgD, and IgG immunoglobulins [68]. Initially Tp0257 was
predicted to be lipid-modified, associated with the outer
membrane and surface exposed, and thus this protein was
supposed to play a role in enabling the T. pallidum to evade
the immune response limiting the antibodies’ cytotoxic and
opsonic capacities (like its homolog in H. influenzae). How-
ever, in further analysis it turned to have a subsurface
localization (like its homolog in E. coli), where substantial
portion of this periplasmic polypeptide is associated with
peptidoglycan layer.
In Brinkman et al. immunoproteome [17] Tp0257 was
reactive with sera from primary, secondary, and early latent
syphilis patients but was not detected by 2DGE immunoblot-
ting method in McGill et al. study [12]. There are few data
about the diagnostic capacities of recombinant Tp0257 in
syphilis serology as an included antigen together with Tp0453
[55].
Tp1038 (TpF1, antigen 4D, antigen C1–5) is homode-
camer comprising 12 identical 19-kDa subunits linked by
disulfide bonds, which form a nearly spherical shell [69]. This
protein plays a role in iron uptake and functionally belongs
to bacterioferritins’ group. Moreover, Tp1038 plays a pivotal
BioMed Research International
role in driving an immune response by activation of inflam-
masome, promoting the development of regulatory T-cells,
modulating the release of specific cytokines by monocytes,
and stimulating an angiogenesis that is typically observed
during secondary syphilis [70].
Tp1038 is seroreactive in Brinkman et al. immunoproteome
[17] study with sera from early latent syphilis, whereas in
McGill et al. study [12] oligomeric form of this antigen exhib-
ited high antibody responses with all stages of syphilis while
it did not exhibit serologic reactivity against the monomeric
form. Currently this antigen has shown a high sensitivity
(93.3–100%) for the detection of all stages of syphilis and
was extremely specific (100%) when tested against potentially
cross-reactive sera, and that proposes Tp1038 to be a promis-
ing candidate for the screening of syphilis [71].
Flagellar proteins form a significant part of T. pallidum
proteome being represented by Tp0868 (FlaB1, 34.5 kDa),
Tp0792 (FlaB2, 33 kDa), and Tp0870 (FlaB3, 31 kDa) filament
core proteins covered with Tp0249 (FlaA1) sheath protein,
also complemented with hook-basal body complex proteins
(Tp0398 and Tp0727) and flagellar motor proteins (Tp0400)
[72]. Now it is hypothesized that Tp0249 (FlaA1) protein is in
a contact with Tp0663 (FlaA2), localized in the inner mem-
brane, and designated as Tromp2 also. These proteins provide
a characteristic corkscrew motility, which is significant for T.
pallidum invasion and dissemination in the host organism.
The hook-basal body complex proteins Tp0398 and
Tp0727 are seroreactive in Brinkman et al. immunoproteome
[17], while in McGill et al. study [12] a number of proteins,
namely, Tp0249, Tp0868, Tp0792, and Tp0870 (FlaA, FlaB1,
FlaB2, and FlaB3, resp.), and flagellar motor protein Tp0400
(FliG), were highly seroreactive at all stages of syphilis. In
early researches some cross-reactions of T. pallidum flagellar
proteins with a number of proteins of distantly related
spirochaetes were observed [73], and that restricted their
diagnostic significance. Recently, a screening of recombi-
nant flagellar proteins showed that FlaB1, FlaB2, and FlaB3
revealed higher overall sensitivity and specificity for IgG
antibody with 95.4% and 98.9%; 92.6% and 95.8%; 95.1%
and 95.8%, respectively [74]. In addition, FlaB1, FlaB2, and
FlaB3 proteins demonstrated an excellent performance for
detecting IgM antibody in primary and congenital syphilis,
with sensitivity and specificity of 76.8% and 83.1%; 72.0% and
87.7%; 74.4% and 89.2%, respectively. These results put FlaB1,
FlaB2, and FlaB3 proteins into the group of novel candidate
antigens for syphilis serodiagnostics.
Thus, current studies (summarized in Table 1) show both
opportunities and limitations of novel recombinant antigens
of T. pallidum for the serological diagnostics of syphilis. Most
typically some of these antigens (Tp0136, Tp0257, and Tp1038)
are more useful for primary and early stages syphilis detec-
tion, while overall they are less sensitive (Tp0155, Tp0483, and
Tp0751) or less specific (Tp0868, Tp0792, and Tp0870) than
conventional immunodominant T. pallidum lipoproteins.
8. Future Directions in Treponema pallidum
Recombinant Proteins Development
The recombinant protein technique can provide a significant
quantity of highly purified T. pallidum antigens for diagnostic
7
use. This led to great progress in a treponema-specific sero-
logical tests reliability based on the detection of antibodies
against T. pallidum immunodominant lipoproteins Tp15,
Tp17, Tp47, and some others. The modern “traditional” and
“reverse” algorithm use this approach as the second- or the
first-line tests, which are effective in most cases of syphilis
diagnostics.
Remaining difficulties in syphilis serological screening
are related to early forms (without expressed immune
response) or late forms of this disease (when immune
response decreases as a result of T. pallidum migration in the
“immunologically privileged” niches). This situation makes it
necessary to recruit new additional treponemal recombinant
antigens that will be seroreactive in cases of ineffectiveness
of immunodominant lipoproteins. For example, development
of surface-exposed proteins Tp0326 (Tp92) and Tp0453 have
increased sensitivity and specificity of serological tests to
98–100%, especially at primary syphilis stage.
Current research studies revealed numerous novel
recombinant antigens, which are promising candidates for
the improved syphilis serological diagnostics. However,
ongoing studies showed the depletion of diagnostic antigens
resource, where some novel products are less effective than
conventional recombinant lipoproteins. The period of rapid
progress in syphilis serodiagnostics development ended and
each new success in this field requires a lot of experimental
and clinical efforts.
On the other hand, the current syphilis laboratory
diagnostics paradigm, which provides alternative diagnostic
result (“yes” or “no”), significantly reduces the experimental
research area, while the immunoproteome studies indicate
greater possibilities for serological analyses compared to
screening and confirmatory tests only. Changes in behavioral
strategy of T. pallidum in the course of the disease determined
by its interaction with the immune system and manifested
in different proteins expression profiles may be a clue for
the differentiation of syphilis stages based on detection of
antibodies against these variable antigens. For example, some
novel T. pallidum recombinant antigens (like Tp0136, Tp0155,
Tp0483, and others) that are not reactive in all syphilis stages
and in current paradigm are assessed as less effective for
syphilis screening are promising candidates for new genera-
tion of immunotests with advanced diagnostic capabilities. In
line with these expectations the quantitative and qualitative
evaluation of antibody level against certain antigens may
reveal a variable immune response (fingerprints) that is
typical for different syphilis stages.
Another unresolved issue is the continued reactivity
of modern conventional treponemal tests for a long time
after the syphilis cure that determines their inefficiency for
monitoring of the treatment response, relapse, or reinfection
in previously treated patients. Currently this goal is achieved
using the low-specific nontreponemal tests, which contrasts
with the experience of the serological monitoring of other
infectious diseases. In this context, the search for novel
T. pallidum antigens, whose antibodies are rapidly eliminated
from host blood flow after the pathogen’s eradication, is
another promising direction in recombinant proteins’ devel-
opment.
 8

Table 1: T. pallidum proteins used and proposed for syphilis serological diagnostics.
Immunoproteomic data
Brinkman et McGill et al. Seroreactivity at Sensitivity/specificity;
Gene (ORF number) Protein name Protein description Links
al. [17] [12] syphilis stages (% of positive result)
Inner membrane lipoproteins
tp0171 Tp15 15 kDa lipoprotein None +++ All stages 100/100 [28]
tp0435 Tp17 17 kDa lipoprotein 9,6–16,6 +++ All stages 96/100 [28]
47 kDa penicillin-
tp0574 Tp47 binding protein, 2,9–10,0 +++ All stages 100/20 [28]
carboxypeptidase
76–100/
tp0768 TmpA 44.5 kDa lipoprotein 8,2–15,3 +++ All stages [32]
99,6
35 kDa lipoprotein, purine
tp0319 TmpC nucleoside receptor A 2,8–6,2 +/++ All stages 100/100 [34]
lipoprotein
38 kDa lipoprotein,
methylgalactoside ABC
Tp38,
tp0684 transporter, 6,8–19,0 +++ All stages ND [35]
MglB-2
galactose/glucose-binding
lipoprotein
32 kDa lipoprotein,
91,0–98,3/
tp0821 Tp32 L-methionine-binding 1,0–1,7 None All stages [38]
94,3–100
lipoprotein
Surface-exposed and outer membrane associated proteins
[43]
Heterogenic antigen
tp0897 TprK None None ND ND [44]
variable by gene conversion
[45]
28-kDa outer membrane
tp0663 TROMP-2 0,8–1,9 None All stages 98,83/100 [47]
protein, FlaA homolog
BamA (𝛽-barrel assembly
86/99 [52]
tp0326 Tp92 machinery protein A) 1,2–2,6 None Mostly at primary
98/97 [55]
ortholog stage; lower reactivity
Proposed carrier of lipids in secondary and 98/100 [52]
tp0453 Tp0453 None +/++
and glycolipids early latent stage 100/100 [55]
BioMed Research International
 Table 1: Continued.
Immunoproteomic data
Brinkman et McGill et al. Seroreactivity at Sensitivity/specificity;
Gene (ORF number) Protein name Protein description Links
al. [17] [12] syphilis stages (% of positive result)
Adhesins
BioMed Research International

Binds to the matrix form of

fibronectin and exhibit low reactive at
tp0155 Tp0155 None None ND (27,9% positive) [55]
peptidase enzymatic primary stage
activity
Binds to both the soluble
Low reactive at
tp0483 Tp0483 and matrix forms of None None ND (41,8% positive) [55]
primary stage
fibronectin
49 kDa outer membrane
tp0136 Tp0136 (lipo)protein; it binds to 0,7–2,1 None Primary stage ND (85,5% positive) [62]
fibronectin and laminin
25,8 kDa protein; it binds to
tp0751 Tp0751 laminin and exhibits None None ND ND (41,8% positive) [55]
metalloprotease activity
Cotranscribed with Tp0751 Primary and early
tp0750 Tp0750 0,8–2,1 None ND [65]
serine protease latent stages
Putative periplasmic proteins
Glycerophosphodiester
phosphodiesterase, binds
tp0257 Gpd Fc-fragment of human IgA, 3,0–7,3 None All stages 91/93 [55]
IgD, and IgG
immunoglobulins
bacterioferritin,
93–100/
tp1038 TpF1, 4D, C1–5 homodecamer from 19-kDa 0,8–2,2 +++ All stages [71]
100
subunits
Flagellar proteins
Flagellar filament 34.5-kDa
tp0868 FlaB1 None +++ All stages 95.4/98.9 [73]
core protein
Flagellar filament 33-kDa
tp0792 FlaB2 None +++ All stages 92.6/95.8 [73]
core protein
Flagellar filament 31-kDa
tp0870 FlaB3 None +++ All stages 95.1/95.8 [73]
core protein
9
10
Thus, the development of enlarged panel of T. pallidum
recombinant antigens remains an actual task of multidisci-
plinary biomedical research. The high-resolution methods
(immunoblot or immunochip) and multicentral examination
protocols allow determining the number of antigens in the
panel with the following criteria: (i) detectable immunore-
activity; (ii) expression level on different syphilis stages; (iii)
decreasing immune response after the infection regress. Scan
of these recombinant antigens and their application for new
aspects of syphilis serological diagnostics are the tasks of the
future research studies.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by a government contract of the
Russian Ministry of Health (Project no. 114/BU-2015-051).
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